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Regular Article |
a Departments of Obstetrics and Gynecology and Physiology and Biophysics, University of Nebraska Medical Center, 984515 Nebraska Medical Center, Omaha, Nebraska 68198-4515
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle, follicular development, growth factors, ovary, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TGF), heparin binding EGF-like growth factor (HB-EGF), amphiregulin, betacellulin, and epiregulin [7]. Although EGFR signaling in most tissues involves the classical MAP-kinase (ERK) [9, 10] pathway, involvement of other signaling pathways, such as protein kinase C [11], phosphatidyl-inositol 3-kinase (PI3-kinase) [12], and G-protein coupled receptors [7, 8] have been documented. The complexity of cellular functions governed by the EGFR and its cross-talk with other key signaling pathways imply that EGFR activity must be precisely regulated during development and dynamic tissue remodeling. Mammalian ovary undergoes tissue remodeling throughout the reproductive life when primary follicles grow to become ovulatory follicles. Therefore, precise temporal recurrence of cell proliferation and differentiation is critical for proper folliculogenesis. Receptor overexpression or underexpression, autocrine ligand stimulation, or constitutively active receptor mutants can cause deregulation of this precisely controlled signaling system, leading to a variety of ovarian pathophysiologies [13], including failure in folliculogenesis. 125I-EGF autoradiography of the rat ovary has demonstrated that EGF-binding sites are present only in the theca interna of large preantral and antral follicles and in granulosa cells of antral follicles [14]. However, the limited resolution of 125I-EGF autoradiography cannot rule out the presence of low levels of EGFR in granulosa cells of small preantral follicles. In fact, primary and secondary follicles of the hamster respond to EGF [4], and EGF phosphorylates a 170-kDa protein in follicular cells, which can be blocked by the tyrosine kinase inhibitor, genistein [15]. FSH stimulates EGF binding in cultured rat granulosa cells [16], and the binding increases following growth hormone treatment [17]. Using 125I-EGF, Moreno-Cuevas et al. [18] have shown that EGFR is present in the rat ovary and the binding increases significantly during estrous.
All these lines of evidence suggest that EGF plays a pivotal role in controlling proliferation and differentiation of granulosa cells during follicular development. However, despite numerous reports about EGF effect and EGFR signaling in ovarian cells [4, 5], the spatiotemporal expression pattern of EGFR messenger RNA (mRNA) and protein in follicular and nonfollicular cells functioning under the endogenous hormonal milieu of the estrous cycle and the selective roles of reproductive hormones in controlling EGFR gene transcription and translation and activation warrant further investigation.
The objectives of the present study were to evaluate systematically the spatiotemporal expression of EGFR mRNA and protein in the ovary throughout the estrous cycle, following the preovulatory gonadotropin surge and after selective endocrine replacement. Further, hormone-induced activation of tyrosine phosphorylation as the initial step in EGFR signaling was also studied. Hamster EGFR cDNA was partially cloned and used as a species-specific nucleic acid probe to study EGFR mRNA expression in ovarian cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-35S]CTP [specific activity, 800 Ci/mmol], was from ICN Radiochemicals (Costa Mesa, CA); and riboprobe synthesis kit was from Promega. All other molecular-grade chemicals were purchased from Sigma (St. Louis, MO), Fisher (Pittsburgh, PA), or United States Biochemical (Cleveland, OH). Ovine-FSH-20 was a gift from the National Pituitary Hormone Program (NIDDK, NIH, Bethesda, MD). Female golden hamsters (90–100 g; SASCO, Madison, WI) with three consecutive estrous cycles were housed under controlled climate and 14L:10D according to the U.S. Department of Agriculture and Institutional Animal Care and Use Committee (IACUC) guidelines. The use of hamsters in this study was approved by the IACUC. Ovaries were obtained during the estrous cycle and following the preovulatory gonadotropin surge and snap frozen on dry ice. To obtain ovaries from hypophysectomized (Hx) hamsters, females were hypophysectomized on Day 1 at 0900 h (estrous), as previously described [19]. Ten days after the Hx, a group of hamsters was injected s.c. either with 10 µg of ovine-FSH-20 twice daily for 2 days, a single dose of 10 I.U. of hCG (Sigma), or a combination of FSH (twice daily for 2 days) and hCG (single injection on the first day) in 0.5% BSA in saline injected at different sites. Control animals received equal volume of vehicle. Ovaries were collected 48 h after the first injection. A second group of Hx hamsters was injected s.c. with a single dose of 0.1 mg of estradiol-valerate (Pharmacia-Upjohn Company, Kalamazoo, MI), 0.5 mg of progesterone (Steraloids, Wilton, NH) in sesame oil (Sigma), a single dose of estradiol injection followed 6 h later by a single dose of progesterone, or equal volume of sesame oil vehicle. Ovaries were collected 24 h after the injection. Ovaries from all groups were snap frozen on dry ice and kept at -80°C until use.
Immunofluorescence Detection of EGF Receptor
Ovaries were sectioned at 6 µm in a Leica (North Central Instruments, Minneapolis, MN) cryostat at -18°C. Sections were fixed in freshly prepared 3% paraformaldehyde in PBS, pH 7.4, at 4°C for 10 min, followed by three 5-min washes in PBS at room temperature [20]. After blocking nonspecific binding sites with PBS containing 10% donkey serum, 0.2% Triton X-100, and 0.1% sodium azide for 30 min in a humidified chamber, sections were exposed overnight to an optimal concentration of EGF receptor (EGFR) antibody at 4°C in a humidified chamber. Sections were then rinsed twice in PBS for 5 min each, followed by a 30-min exposure to goat-anti-rabbit-Alexa-488 (green fluorescence) in a humidified chamber at room temperature. The nuclei were stained with propidium iodide (red fluorescence) to visualize nuclear localization of EGFR, which retained its green fluorescence after image merger, and to define follicle structure. The specificity of the antibody was verified by incubating ovarian sections without primary antibodies. After thorough rinsing, sections were mounted with Fluoromount G (Southern Biotechnology, Birmingham, AL) and evaluated under epifluorescence in a Leica DMR research microscope equipped with an Optronics Magnafire digital camera.
For digital image capturing, the exposure time was adjusted using sections incubated without the primary antibody to minimize any auto or nonspecific fluorescence recording without compromising actual signal. The signal obtained after such a background correction was considered an antigen-specific signal. For each image, EGFR staining (green) was merged with nuclear staining (red) using Magnafire software that caused virtually no pixel shifting during image merger and resulted in shades of red to reddish yellow to reddish green to green. Immunofluorescence localization studies were repeated at least three times using tissues from different animals to verify the reproducibility of the data. Representative photomicrographs were arranged using Adobe Photoshop (San Jose, CA) without any further adjustment to maintain the true nature of the findings. The intensity of immunosignal was assessed from an intensity scale in which highest and lowest intensities were indicated by 4+ and ±, respectively. There were 
4 replicates for each type of follicle and thecal and interstitial cells from each section, and there were three different sections from three animals for each group. Preantral follicles at stages 5 and 6 and antral follicles regardless of the size were used for quantification.
Reverse Transcription-PCR Cloning of Hamster EGFR cDNA
Because no nucleic acid information about hamster EGFR was available in the Gene Bank, we partially cloned hamster EGFR cDNA to obtain species-specific nucleic acid probes to study ER mRNA expression in hamster ovarian cells. The sequences of primer pairs for generating hamster EGFR cDNA were selected after comparing human [21], rat [22], and mouse [23] EGFR cDNA sequences. PCR was done in two stages to obtain hamster-specific EGFR cDNA. The sequences of the forward and reverse primers for the first stage of PCR were 5'-GCTGTCCCAATGGAAGCTGCTGG-3' and 5'-GCCACTTGGCAGGATGTG-3', respectively. Poly(A)+ RNA from proestrous hamster ovaries was prepared as described previously [24] and reverse transcribed using gene-specific reverse primer to generate EGFR cDNA, which was denatured at 94°C for 4 min and amplified for 30 cycles in an MJ Research (Waltham, MA) thermocycler using the following conditions: 1 min at 94°C, 1 min at 45°C, 1 min at 72°C, followed by a 10-min extension at 72°C. The final Mg2+ concentration was 2.8 mM. Because 100% complementarity was not expected between primers and hamster EGFR mRNA, an annealing temperature of low stringency was selected to allow maximum hybridization. A major product of a predicted 547 base pair (bp) was obtained following PCR amplification. The cDNA was extracted from the gel, sequenced in an automated DNA sequencer (University of Nebraska Medical Center's Genetic Sequence Core), and compared with EGFR sequences of other species (Gene Bank). A second pair of primers was synthesized based on the hamster EGFR cDNA sequence for nested PCR of EGFR in the second stage. The sequences of the forward and reverse primers for the nested EGFR PCR were 5'-GAGTGACTGTCTGGTCTG-3' and 5'-CGCCATCTTCTTCCAC-3', respectively. For nested EGFR PCR, hamster ovarian poly(A)+ RNA was reverse transcribed in the presence of nested reverse primer, and the resulting cDNA was denatured at 94°C for 4 min and amplified using the following conditions: 1 min at 94°C, 1 min at 55°C, 1 min at 72°C, followed by a 10-min extension at 72°C. A single cDNA of 233 bp was obtained following 30 cycles of PCR. The cDNA was inserted in PGEM-T easy plasmid and transformed in JEM109 cells (Promega). The plasmid was purified using Qiagen (Valencia, CA) plasmid Maxi kit according to the manufacturer's protocol and was analyzed for the presence of hamster EGFR cDNA by PCR, restriction digestion, and sequencing. Plasmid DNA with the EGFR cDNA insert was linearized for riboprobe synthesis.
Northern Hybridization Detection of Hamster EGFR in the Ovary
Northern hybridization of hamster EGFR was done essentially as described by Roy [24] and Yang et al. [25]. Poly(A)+ RNA was prepared from proestrous (Day 4, 0900 h) hamster ovaries, and 2.5 µg was fractionated in a denaturing formaldehyde gel, transferred to Nytran membrane (Schleicher and Schuell), and UV cross-linked. The membrane was stained briefly with methylene blue to mark the positions of the RNA size ladder, hybridized overnight with 32P-antisense or sense cRNA at 50°C in the presence of 40% formamide, washed stringently, and exposed to Kodak x-ray film for 48 h. Approximate RNA size was determined from the positions of the RNA size markers (Gibco-BRL, Carlsbad, CA).
In Situ Hybridization Localization of Spatiotemporal Expression of EGFR mRNA in Hamster Ovarian Cells
In situ hybridization was done as described by Yang et al. [25], with some modifications. Briefly, frozen sections of ovaries were dried on a hot plate at 45°C and fixed in 4% fresh paraformaldehyde in PBS, pH 7.4, on ice for 10 min, followed by three 10-min rinses with ice-cold PBS. After acetylation for 10 min, sections were rinsed in 4x saline-sodium citrate (SSC) and dehydrated through ascending grades of ethanol at room temperature. Sections were prehybridized in 50% formamide for 30 min at 37°C, followed by a 4-h hybridization in 6x SSC, 10 mM NaH2PO4, 50 µg/ml yeast tRNA, 1% dextran sulfate, 50% formamide, 1 mM dithiothreitol (DTT), and 2 x 107 counts per minute 35S-antisense or sense cRNA per ml at 45°C in a humidified chamber. After thorough rinsing in 4x SSC, nonhybridized probe was removed by RNaseA digestion, followed by rinsing in 1x SSC for 30 min and dehydration in ascending grades of ethanol. Sections were finally coated with NTB2 nuclear track emulsion diluted 1:1 with 0.3 M Na-acetate, dried, exposed in the dark for the optimum time, developed in 1:1 Dektol (Kodak) in water, stained with hematoxylin and eosin, and mounted in DPX (BDH, Poole, England). All sections were evaluated under bright- as well as dark-field illumination for silver grain distribution as an index of mRNA expression. Finally, in situ signal intensity in granulosa cells of preantral and antral follicles and in thecal and interstitial cells was quantified using NIH Image version 1.6 image analysis software. Signal intensity recorded from corresponding sections that were hybridized with the sense probe was subtracted from the total signal intensity for each cell type to obtain specific signal intensity. There were 4 replicates for each type of follicle and thecal and interstitial cells from each section, and there were two different sections from two animals for each group. Preantral follicles at stages 5 and 6 and antral follicles regardless of the size were used for quantification. No attempt was made to quantify small preantral follicles because we could not identify adequate numbers with morphological certainty for statistical analysis. The results were expressed as optical density per pixel.
Immunolocalization studies were repeated at least three times, while in situ hybridization was repeated twice to insure reproducibility and to verify statistical significance whenever appropriate.
Detection of Steady-State Levels of Ovarian EGFR mRNA During the Estrous Cycle and Following Hormonal Manipulations
The steady-state levels of ovarian EGFR were evaluated by semiquantitative reverse transcription-PCR (RT-PCR) essentially as described by Roy [24] and Yang et al. [25] using total ovarian RNA and random hexamers for cDNA synthesis and nested EGFR forward and reverse primers as described earlier. The PCR conditions optimized during cloning were used during the assay. The steady-state levels of ovarian ß-actin mRNA were also evaluated to determine the specificity of changes in EGFR mRNA levels. The levels of ß-actin mRNA in the adult hamster ovary remained steady throughout the cycle as well as after hormonal manipulations. The forward and reverse primers for hamster ß-actin were 5'-GGGCCAGAAGGACTCGTACG-3' and 5'-CACAGCTTCTCTTTGATGTCACGCAC-3', respectively, and were designed from a hamster ß-actin cDNA sequence (accession number AJ312092, 141–650 bp) from the NCBI Gene Bank. The PCR conditions for ß-actin were identical to EGFR, and the expected size of the amplicon was 510 bp, which was verified further by sequencing. The PCR products were fractionated in a 1.5% agarose gel, transferred to Nytran membrane, and Southern hybridized with respective 32P end-labeled gene-specific oligodeoxynucleotides probes as described previously [24]. The radioactive signal was quantified as digital light units (DLUs) in a Cyclone Phosphorimager (Packard Instrument Company, Meriden, CT) and normalized against the ß-actin signal. The results were expressed as DLU EGFR mRNA relative to ß-actin. There were three replicates for each group.
Detection of Protein-Tyrosine Phosphorylation in Ovarian Cells Following Endogenous and Exogenous Gonadotropin Exposure
Protein tyrosine phosphorylation, as an index of the first step in EGFR activation in ovarian cells, was evaluated in order to delineate whether higher EGFR expression correlated with increased EGFR activation and whether gonadotropin action leads to EGFR activation. Because none of the commercial phospho EGFR antibodies was useful to detect activated EGFR in the hamster ovary, we evaluated ovarian EGFR activation in situ by phosphotyrosine antibody and, using AG1478, an EGFR-specific tyrosine kinase inhibitor. Autophosphorylation of tyrosine residues of the receptor endodomain was the first rate-limiting step in EGFR activation, downstream of ligand binding and receptor dimerization, and EGFR activation is a major tyrosine phosphorylation event in cells. Therefore, at least a part of the phosphorylation signal would reflect activated EGFR. For positive control, female hamsters were injected with a single dose of 10 µg of murine EGF (Collaborative Research, Bedford, MA) i.p. on Day 4 at 0900 h, and ovaries were collected 2 h later and snap frozen on dry ice. Ovaries from cyclic hamsters at Day 4 (0900 and 1600 h) were used to study the effect of endogenous gonadotropins. Ovaries from hypophysectomized hamsters treated with vehicle, 10 µg ovine-FSH (NIH-20) twice daily for 2 days, or 10 µg ovine-FSH twice daily for 2 days plus a single injection of 10 I.U. hCG on the first day were used to evaluate the selective role of FSH in EGFR activation. Fresh, frozen sections were fixed in freshly prepared ice-cold 1% paraformaldehyde in PBS, pH 7.4, for 10 min, followed by the staining protocol described earlier. Nuclei were counterstained with propidium iodide to identify the follicular structure.
In a separate experiment, 5 µl of a 60 nM solution of AG1478 in saline was injected into the ovarian bursa of proestrous hamsters at 1200 h, i.e., 2 h before the preovulatory gonadotropin surge. Ovaries were collected at 1600 h and processed for immunofluorescence detection of tyrosine-phosphorylated protein.
Immunolocalization studies were repeated at least three times, while in situ hybridization was repeated twice to insure reproducibility. All quantitative data were analyzed by ANOVA and the Scheffe F-test (StatView; Abacus Concepts, Berkeley, CA) to determine the level of significance (P < 0.05).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Overall, EGFR immunoreactivity was comparatively more in theca and interstitial cells than granulosa cells; however, EGFR expression in granulosa cells depended on the stage of follicular development. Table 1 summarizes the differential expression of EGFR in various ovarian compartments during the estrous cycle and after hormone administration in hypophysectomized hamsters. On estrous morning (Day 1, 0900 h), a distinct EGFR signal was present primarily in theca cells surrounding large preantral follicles (stage 6: 7–8 layers of granulosa cells [26]); interstitial cells had modest immunosignal (Fig. 1A). EGFR immunoreactivity increased markedly in theca and interstitial cells by Day 3 (0900 h) of the estrous cycle (Fig. 1C). Granulosa cells of small preantral as well as of primordial follicles showed modest but noticeable immunoreactivity (Fig. 1C, insert). Most notably, distinct EGFR immunoreactivity was present in the nucleus of the oocytes (arrowhead), regardless of the stage of follicle development, and in granulosa cells (Fig. 1C, inserts, arrowhead). The increase in EGFR expression was prominent in granulosa cells of large preantral (stage 6) and small antral follicles (stage 8; Fig. 1C). EGFR immunoreactivity increased markedly in the granulosa cells of large antral follicles (stage 10; Fig. 1D) on proestrous (Day 4, 0900 h) but remained relatively steady in preantral follicles and follicles with incipient antrum (stage 7 [26]; Fig. 1D). EGFR protein expression in theca, interstitial, and surface epithelial cells remained high following the preovulatory gonadotropin surge (Day 4, 1600 h; Fig. 1E); however, mural granulosa cells appeared to have stronger EGFR immunosignal compared with antral granulosa cells and theca cells (Fig. 1E). No EGFR-specific signal could be detected when sections of Day 4 (1600 h) ovaries were incubated without the primary antibody (Fig. 1B), suggesting the specificity of the immunosignal.
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EGFR immunoreactivity was virtually absent in ovarian cells following the removal of the pituitary gonadotropins (Fig. 2A). The hCG stimulated noticeable increase in EGFR expression primarily in the thecal and interstitial cells present adjacent to the preantral follicles (Fig. 2B). In contrast with the hCG effect, FSH replacement for 2 days resulted in follicular development up to antral stages concurrent with a marked increase in EGFR immunoreactivity in granulosa of large preantral (stage 6) and antral follicles as well as in interstitial cells (Fig. 2C). A distinct EGFR immunosignal was located in the oocyte (Fig. 2C) as well as in the nuclei of granulosa cells (Fig. 2C, arrowheads). Similar to that observed during the preovulatory phase of the estrous cycle, EGFR expression in antral follicles was preferentially more in mural than antral granulosa cells (Fig. 2C). Theca cells, on the other hand, showed a very low level of EGFR expression (Fig. 2C). Estradiol caused a noticeable increase in EGFR expression in granulosa cells of preantral follicles, but an increase also occurred in the oocyte (Fig. 2D). Nuclear localization of EGFR in the interstitial cells was also prominent following estradiol administration (Fig. 2D). Whereas progesterone failed to induce EGFR expression in granulosa cells of preantral follicles, it markedly stimulated EGFR expression in thecal (primarily) as well as in interstitial cells (Fig. 2E).
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EGFR mRNA Expression Levels in the Hamster Ovary and the Effects of Hormones
Northern hybridization revealed three distinct EGFR transcripts of >6.58, 
4.0, and 2.0 kb in hamster ovarian poly(A)+ RNA (Fig. 3). No signal was obtained when membrane was probed with 32P-labeled sense probe (data not shown), indicating the specificity of the hybridization. Sequence comparison revealed that hamster EGFR cDNA was 96%, 93%, 79%, 54%, and 50% identical to the corresponding regions of the mouse [23], human [21], and rat [22] EGFR cDNA, respectively.
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The intensity of in situ hybridization signal is presented in Figure 4. In situ hybridization analysis revealed that, on Day 1 of the estrous cycle, EGFR mRNA expression was limited primarily to theca and interstitial cells, while EGFR transcript levels in granulosa cells were modest (Figs. 4 and 5A). EGFR mRNA expression increased considerably on Day 3 (0900 h) in theca and granulosa cells of antral follicles as well as in the interstitial cells (Figs. 4 and 5C); however, granulosa cells (GC) of preantral follicles showed no significant (P < 0.05) increase (Figs. 4 and 5C). Mural GC of atretic follicles showed modest expression (Fig. 5C, signal not quantified). EGFR mRNA expression in granulosa cells of antral follicles, theca, and interstitial cells continued to increase through Day 4 (Figs. 4 and 5D); however, a small but significant increase in EGFR mRNA signal occurred in granulosa cells of preantral follicles compared with Day 1 (0900 h) (Figs. 4 [insert] and 5D). EGFR mRNA expression was also noted in cells of the surface epithelium (Fig. 5, D and E). A further increase in EGFR mRNA levels occurred in all cell types following the gonadotropin surge (Figs. 4 and 5E). Notably, receptor mRNA expression was more pronounced in granulosa cells of large preantral (stage 6) and early antral follicles (stage 7; Figs. 4 and 5E) and their adjacent thecal layers. It was noteworthy that the level of EGFR mRNA expression was more in mural granulosa cells relative to antral granulosa cells and in interstitial cells adjacent to follicles compared with those present elsewhere (Fig. 5E). No receptor-specific mRNA was localized when hybridization was done in the presence of 35S-labeled sense probe, indicating the specificity of the in situ data (Fig. 5B).
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The intensity of the in situ hybridization signal is presented in Figure 6. Hypophysectomy resulted in a significant decrease in EGFR mRNA levels in granulosa as well as in thecal cells (Figs. 6 and 7A). EGFR mRNA expression increased significantly in granulosa cells of preantral and antral follicles, and theca and interstitial cells coincided with follicular development in the ovaries of FSH-treated hypophysectomized hamsters (Figs. 6 and 7B). A clear-cut difference in the level of receptor mRNA expression was evident for mural versus antral granulosa cells (Figs. 5, C–D, and 6). Interestingly, interstitial cells adjacent to growing follicles had maximum EGFR mRNA expression compared with their location elsewhere in the ovary (Fig. 7B). In agreement with the immunofluorescence data, hCG stimulated EGFR mRNA expression primarily in theca and interstitial cells (Figs. 6 and 8A). Whereas a significant increase in EGFR mRNA expression occurred in theca, granulosa, and interstitial cells following estradiol treatment (Figs. 6 and 8B), the progesterone effect was limited to theca and interstitial cells (Figs. 6 and 8C). Interestingly, progesterone strongly attenuated the estradiol effect on EGFR mRNA levels in all cell compartments in the ovary (Figs. 6 and 7D).
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The levels of EGFR mRNA were low on Day 1 (0900 h) but increased steadily up to Day 4 (0900 h) (Fig. 9A). A marked increase in the mRNA levels was evident following the preovulatory gonadotropin surge (Fig. 9A). Whereas EGFR mRNA expression decreased significantly (P < 0.05) following hypophysectomy (Fig. 9B), maximum induction in EGFR gene expression occurred following FSH and estrogen replacement (Fig. 9B). Although a considerable increase in EGFR mRNA levels was also noted following hCG or progesterone replacement (Fig. 9B), progesterone significantly attenuated the stimulatory effect of estrogen (Fig. 9B).
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In Vivo Activation of Ovarian EGF Receptor
Induction of protein tyrosine phosphorylation was detected on Day 4 (0900 h) in granulosa and thecal cells as punctate discrete staining (Fig. 10A). Further, the signal was more pronounced in mural than antral granulosa cells of large antral follicles (Fig. 10A). However, the intensity of staining in mural GC or theca of small antral follicles was relatively low (Fig. 10A). EGF administration resulted in a significant induction of tyrosine phosphorylation primarily in mural granulosa cells (Fig. 10B), which also expressed high levels of EGFR (Fig. 1D). Interestingly, phosphotyrosine signals in theca cells did not change appreciably (Fig. 10B), even though these cells constitutively expressed high levels of EGFR (Fig. 1D). Similar to exogenous EGF, the preovulatory gonadotropin surge also resulted in an impressive protein tyrosine phosphorylation of granulosa cells of antral follicles, and the intensity of the signal was far more than that observed with EGF (Fig. 10C). In contrast with the selective EGF effect, the punctate phosphotyrosine signal was located in interstitial and thecal cells (Fig. 10C) following the gonadotropin surge. A distinct but low level of phosphotyrosine signal was detected primarily in the zona pellucida of preantral follicles, through which granulosa cell processes traverse to be in contact with the oocyte (Fig. 10C). The preovulatory gonadotropin surge-induced protein tyrosine phosphorylation was markedly attenuated in GC and theca of large as well as small antral follicles following the administration of AG1478 (Fig. 10D). Further, AG1478 inhibited the basal tyrosine phosphorylation, as evident from the low immunosignal present in the thecal cells (Fig. 10D). A phosphotyrosine signal was virtually absent in granulosa, theca, and interstitial cells in ovaries of hypophysectomized hamster (Fig. 10E); however, a significant increase in protein tyrosine phosphorylation occurred in granulosa cells, primarily in mural layers, of antral follicles developed under the influence of exogenous FSH (Fig. 10G). A small but noticeable increase was also noted in thecal cells (Fig. 10G). No phosphotyrosine signal could be detected in large antral follicles on Day 4 (1600 h) in the absence of primary antibody, indicating the specificity of the immunosignal (Fig. 10F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of EGFR in follicular cells has been demonstrated by localization [3, 14, 27] and functional [4, 28–33] studies. Chabot et al. [14] have demonstrated that maximum EGF binding occurs in theca and luteal cells. Whereas low binding occurs in granulosa cells of growing follicles, a significant increase in ligand binding is evident in granulosa cells of preovulatory follicles [14]. Our results correlate well with their findings and provide additional information about the spatiotemporal expression pattern of EGFR protein and mRNA in follicular and nonfollicular cells during dynamic changes in gonadotropins and ovarian steroid hormones throughout the estrous cycle. The expression of EGFR protein correlates well with the expression of EGFR mRNA, suggesting that gonadotropins and steroid hormones influence EGFR gene transcription and translation in the ovary. In the hamster, EGF immunoreactivity in granulosa cells inversely correlates with follicle development [2] during the estrous cycle, and preantral follicles produce EGF in response to FSH [34]. These findings together with the present results suggest that EGFR presentation forms a key regulator of EGF action in follicular cells. Higher levels of EGFR in antral follicles cannot be attributed to a lack of ligand-mediated down-regulation of the receptor because high protein levels coincide with increased levels of receptor mRNA, and both are stimulated by FSH.
EGFR controls a diverse range of cellular functions, such as cell division, cell-fate decisions, and differentiation in vertebrate as well as in invertebrates [34–36]. The presence of low levels of EGFR protein in granulosa cells of small preantral follicles may be necessary for transducing a downstream signal that is adequate for cell division. On the other hand, higher expression of EGFR may lead to activation of additional signal pathways critical for controlling the differentiation functions of ovarian cells. EGFR can activate multiple different signal pathways regulating cell survival and differentiation [8, 36, 37]. An enhanced rate of transcription and translation of EGFR in granulosa cells of antral follicles also suggests that maturation of granulosa cells is a key determinant of stage-specific induction of EGFR, which is regulated by a combined action of FSH and LH.
Mondschein and Schomberg [38] have shown that EGF attenuates a FSH-stimulated increase in 125I-LH binding to cultured granulosa cells of diethylstilbestrol-treated immature rats. The low levels of EGFR transcription and translation in preantral follicles agree with their results for proliferative granulosa cells. EGF is a major mitogen for hamster granulosa cells [6, 34] and LH receptor does not appear in granulosa cells until follicles reach the antral stage [4, 39, 40]. It may be speculated that higher EGFR signaling via receptor over expression is linked to granulosa cell differentiation and progesterone production in vivo. EGF significantly stimulates progesterone production by hamster antral follicles in vitro [41]. Pestell et al. [42] have demonstrated that EGF significantly (fourfold) stimulates CYP11A1 (cholesterol side chain cleavage enzyme) promoter by a ras/MEK1/Ap-1-dependent mechanism. Increased levels of tyrosine phosphorylation in mural granulosa cells relative to antral granulosa cells and a significant increase in tyrosine phosphorylation following the gonadotropin surge suggest that LH-induced terminal differentiation of granulosa cells involves increased tyrosine kinase signaling. The exact reasons for the low phosphotyrosine signal, despite high levels of EGFR expression in theca cells, are not known at present; however, it is likely that activation of EGFR in theca and interstitial cells may be a continuous process; hence, no significant activation can be evoked by the gonadotropin surge or even by EGF. Attenuation of gonadotropin surge-induced tyrosine phosphorylation by AG1478 provides strong evidence in support of EGFR activation. FSH induction of EGFR synthesis as well as activation are evident from the results of FSH-treated Hx hamsters. FSH stimulates follicular EGF production in the hamster both in vivo [2] and in vitro [34]. Further, antibody to EGF blocks FSH-induced follicular [3H]thymidine incorporation in vitro [6]. Because preantral follicles gradually develop complex steroidogenic capacity as they grow through higher stages, increased expression of EGFR may occur under the influence of FSH and the estradiol milieu of the estrous cycle.
Higher expression of EGFR protein and mRNA in presumptive thecal cells in the hamster ovary and its further increase concurrent with thecal development suggests increased EGFR signaling may be needed for interstitial to thecal cell transition. This is in conflict with Erickson and Case [43], who have reported that EGF inhibits rat theca-interstitial cytodifferentiation in vitro. This discrepancy may be due either to species or to their use of in vitro cell culture that does not completely mimic in vivo conditions. The relatively higher expression of EGFR protein and mRNA in interstitial cells immediately outside the granulosa cell compartment may be mediated by estrogen produced by these follicles. Morphologically distinct thecal cells do not appear in hamster preantral follicles until they reach stage 5 (5–6 layers of granulosa cells [26]), and follicular ability to produce estradiol from androgen precursor is evident from stage 3 onward [41]. The strong attenuation of the estrogen-induced increase in EGFR mRNA in granulosa, thecal, and interstitial cells by progesterone suggests that proliferative and differentiation functions of ovarian cells are modulated by a critical interplay of these two main ovarian steroids. Progesterone strongly inhibits estrogen receptor mRNA and protein levels in the hamster ovary [23].
The presence of EGFR mRNA and protein in the oocyte correlates well with EGF-induced maturation of follicle-enclosed rat oocytes [44] and mouse oocytes in vitro [45]. Further, present results demonstrate for the first time a clear-cut presence of nuclear EGFR both in granulosa cells and the oocyte, suggesting that, besides activation of multiple signaling cascade(s) [8], EGFR may function as a transactivation factor. The distinct nuclear presence of EGFR has been observed in regenerating liver [46] and in a variety of cancer cells [47–49]. Although we do not know at present whether nuclear EGFR represents intact receptor protein, there is no evidence so far to suggest that degraded membrane receptor cycles through the nucleus. Recently, Lin et al. [50] have elegantly demonstrated that the carboxy terminus of EGFR contains a strong transactivation domain, binds cyclin D1 promoter both in vitro and in vivo, and activates cyclin D1 promoter in vitro, thus providing a direct link between nuclear EGFR and proliferating activities of cells.
In summary, EGFR mRNA and protein are expressed in hamster ovarian cells in a spatiotemporal fashion and the expression is differentially modulated by gonadotropins, estrogen, and progesterone. Further, EGFR activation in the ovary may be related not only to cell division but also to postmitotic differentiation depending on the degree of receptor expression. Therefore, a regulated expression of EGFR, hence, activation, may be a key to direct ovarian cells from proliferative to differentiation phenotypes. If higher a EGFR signal is necessary for granulosa cell differentiation, an internal switch must be activated during preantral to antral transition to trigger enhanced EGFR transcription and translation.
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FOOTNOTES |
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2 Correspondence. FAX: 402 559 6164; skroy{at}unmc.edu
Received: 13 March 2002.
First decision: 4 April 2002.
Accepted: 5 June 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER-) and estrogen receptor-ß (ER-ß) in the hamster ovary: differential regulation by gonadotropins and ovarian steroid hormones. Endocrinology 2002; (in press) This article has been cited by other articles:
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  M. Zhang, H. Ouyang, and G. Xia The signal pathway of gonadotrophins-induced mammalian oocyte meiotic resumption Mol. Hum. Reprod., July 1, 2009; 15(7): 399 - 409. [Abstract] [Full Text] [PDF]
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 J.J.H. Celestino, J.B. Bruno, I.B. Lima-Verde, M.H.T. Matos, M.V.A. Saraiva, R.N. Chaves, F.S. Martins, L.F. Lima, K.P.O. Name, C.C. Campello, et al. Recombinant Epidermal Growth Factor Maintains Follicular Ultrastructure and Promotes the Transition to Primary Follicles in Caprine Ovarian Tissue Cultured In Vitro Reproductive Sciences, March 1, 2009; 16(3): 239 - 246. [Abstract] [PDF]
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 I. Ben-Ami, L. Armon, S. Freimann, D. Strassburger, R. Ron-El, and A. Amsterdam EGF-like growth factors as LH mediators in the human corpus luteum Hum. Reprod., January 1, 2009; 24(1): 176 - 184. [Abstract] [Full Text] [PDF]
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K.N. Fru, M. Cherian-Shaw, M. Puttabyatappa, C.A. VandeVoort, and C.L. Chaffin Regulation of granulosa cell proliferation and EGF-like ligands during the periovulatory interval in monkeys Hum. Reprod., May 1, 2007; 22(5): 1247 - 1252. [Abstract] [Full Text] [PDF]
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 P. Yang and S. K. Roy A Novel Mechanism of FSH Regulation of DNA Synthesis in the Granulosa Cells of Hamster Preantral Follicles: Involvement of a Protein Kinase C-Mediated MAP Kinase 3/1 Self-Activation Loop Biol Reprod, July 1, 2006; 75(1): 149 - 157. [Abstract] [Full Text] [PDF]
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P. Yang and S. K. Roy Transforming Growth Factor B1 Stimulated DNA Synthesis in the Granulosa Cells of Preantral Follicles: Negative Interaction with Epidermal Growth Factor Biol Reprod, July 1, 2006; 75(1): 140 - 148. [Abstract] [Full Text] [PDF]
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 Z. Zhao, D. Garbett, J. L Hill, and D. J Gross Epidermal growth factor receptor downregulation in cultured bovine cumulus cells: reconstitution of calcium signaling and stimulated membrane permeabilization Reproduction, October 1, 2005; 130(4): 517 - 528. [Abstract] [Full Text] [PDF]
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 J.-H. Choi, K.-C. Choi, N. Auersperg, and P. C K Leung Gonadotropins upregulate the epidermal growth factor receptor through activation of mitogen-activated protein kinases and phosphatidyl-inositol-3-kinase in human ovarian surface epithelial cells Endocr. Relat. Cancer, June 1, 2005; 12(2): 407 - 421. [Abstract] [Full Text] [PDF]
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 P. Yang, J. Wang, Y. Shen, and S. K. Roy Developmental Expression of Estrogen Receptor (ER) {alpha} and ER{beta} in the Hamster Ovary: Regulation by Follicle-Stimulating Hormone Endocrinology, December 1, 2004; 145(12): 5757 - 5766. [Abstract] [Full Text] [PDF]
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